hTERT启动子调控的异种抗原基因α-1,3GT表达载体的构建及其在肺癌细胞中的靶向表达

Construction of xenoantigen α-1,3GT gene expression vector regulated by hTERT promoter and its targeting expression in human lung cancer cells

目的:构建人端粒酶逆转录酶(human telomerase reverse transcriptase,hTERT)启动子调控的异种移植抗原α-1,3半乳糖基转移酶(α-1,3galactosyltransferase,α-1,3GT)基因真核表达载体,研究其调控的α-1,3GT在肿瘤细胞中的靶向表达。方法:将前期克隆并测序正确的猪α-1,3GT基因定向克隆到pEGFP-hTERTp质粒中,构建α-1,3GT真核表达载体pEGFP-hTERTp-GT。分别将pEGFP-hTERTp-GT和CMV启动子调控的α-1,3GT真核表达质粒pEGFP-N1-GT转染端粒酶阳性的人肺癌细胞A549及端粒酶阴性的正常人胚肺成纤维细胞MRC-5。RT-PCR检测转染细胞中α-1,3GTmRNA的表达,免疫荧光法和流式细胞仪检测α-gal抗原的表达。结果:成功构建了pEGFP-hTERTp-GT真核表达载体。转染pEGFP-N1-GT的A549和MRC-5中均有α-1,3GTmRNA的表达;转染pEGFP-hTERTp-GT的A549中有α-1,3GTmRNA的表达,而端粒酶阴性的MRC-5细胞中无α-1,3GTmRNA的表达。转染pEGFP-N1-GT的A549和MRC-5中均可表达异种移植抗原α-gal;转染pEGFP-hTER-Tp-GT质粒的A549中有α-gal的表达,而MRC-5细胞中无α-gal的表达(P<0.01)。结论:hTERT启动子调控的α-1,3GT基因能靶向性表达在端粒酶阳性的肺癌细胞中,并合成异种移植抗原α-gal。

Objective：To construct an xenoantigen synthetase α-1,3 galactosyltransferase （α-1,3GT） eukaryotic expression vector regulated by human telomerase catalytic subunit （hTERT） promoter, and to investigate its targeting expression of α-1,3GT in lung cancer cell lines. Methods： Previously prepared and confirmed pig α-1,3GT gene was inserted into pEGFPhTERTp plasmid to construct eukaryotic expression vector pEGFPhTERTpGT. pEGFPhTERTpGT and pEGFPN1GT （α1,3GT eukaryotic expression vector under the control of CMV promoter） were transfected into telomerasepositive human lung adenocarcinoma A549 cells and telomerasenegative human embryonic lung fibroblast MRC5 cells. 1,3GT mRNA expression in the transfected cells was detected by RTPCR. Expression of αgal antigen in transfected cells was examined by immunofluorescence and flow cytometry. Results： pEGFPhTERTpGT eukaryotic expression vector was successfully constructed. Both A549 and MRC5 cells transfected with pEGFPN1GT showed expression of α-1,3GTmRNA; A549 cells but not telomerasenegative MRC5 cells expressed α-1,3GT mRNA after transfection with pEGFPhTERTpGT. Furthermore, both A549 and MRC5 cells transfected with pEGFPN1GT showed expression of xenoantigen α-gal; A549 but not MRC5 cells expressed xenoantigen αgal after transfection with pEGFPhTERTpGT （P〈0.01）. Conclusion： α1,3GT gene under the regulation of hTERT promoter can be specifically expressed in telomerasepositive lung cancer cell lines, which can induce production of xenoantigen α-gal.